Irina Yurkova

Radiation-induced peroxidation and fragmentation of lipids in a model membrane

Purpose : To ascertain the possibility of fragmentation processes in lipid membranes when acted upon with γ-radiation under various conditions, and to evaluate fragmentation processes quantitatively in comparison with the lipid peroxidation processes. Materials and methods : Phospholipids as components of multilamellar liposomes exposed to 137 Cs γ-rays at dose-rates of 0.06 and 0.33 Gy s -1 in the dose range 0-18 kGy. Peroxidation products were determined spectrophotometrically and fragmentation products were analysed using thin-layer chromatography and GLC methods. Results : Phosphatidylglycerol containing unsaturated fatty acid residues and free hydroxyl groups underwent both peroxidation and fragmentation when its aqueous dispersions were treated with ionizing radiation. γ-Radiolysis of multilamellar liposomes led to the formation of peroxidation products, as well as phosphatidic acid and hydroxyacetone, fragmentation products of the initial lipid. Comparable radiation-chemical yields of conjugated-diene products and phosphatidic acid indicate that the probabilities of oxidation and fragmentation processes to occur are approximately equal in this case. In contrast to lipid peroxidation, lipid fragmentation is not accelerated but suppressed by oxygen and appears to be a prevailing process in de-aerated solutions. The phosphatidic acid formed as a result of phosphatidylglycerol fragmentation can intensify the lipid peroxidation. Conclusions : Treatment of lipid membranes with ionizing radiation leads to the destructive processes within both hydrophobic and hydrophilic moiety of lipids.

Quinones as free-radical fragmentation inhibitors in biologically important molecules

Effects of a number of quinones and diphenols of various structures on free-radical fragmentation processes taking place in f -diols, glycerol, 2-aminoethanol, glycero-1-phosphate, ethylene glycol monobutyrate, maltose, and some lipids were investigated. Quinone additions have been found to change the direction of free-radical transformations of the compounds cited above by inhibiting formation of the respective fragmentation products owing to oxidation of radicals of the starting compounds. The results obtained and literature data available allow a suggestion to be made that the system quinone/diphenol is able to not only deactivate or generate such active species as O 2 ” m but also control the realization probability of free-radical processes of peroxidation and fragmentation in biologically important molecules.

Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b−/− mouse model of Wilson's disease

Cellular copper overload as found in Wilsons disease may disturb mitochondrial function and integrity. Atp7b(-/-) mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu(2+)/H(2)O(2)/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b(-/-) mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b(-/-) mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b(-/-) mice in contrast to control with increasing age. Hepatocytes from elder Atp7b(-/-)mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilsons disease.

Formation of phosphatidic acid in stressed mitochondria.

Mitochondria are an important intracellular source of ROS as well as a sensitive target for oxidative damage under certain pathological conditions such as iron or copper overload. Mitochondrial membranes are rich in the tetraacyl phospholipid cardiolipin. Its integrity is important for efficient oxidative phosphorylation. Mouse liver mitochondria were subjected to oxidative stress by the Cu(2+)(Fe(2+))/H(2)O(2)/ascorbate system. Phosphatidic acid was detected in oxidized mitochondria, but not in unperturbed mitochondria. The Cu(2+)/H(2)O(2)/and (or not) ascorbate system caused the formation of phosphatidic acid and phosphatidylhydroxyacetone in cardiolipin liposomes. These products proceed via an HO*-radical induced fragmentation taking place in the polar moiety of cardiolipin. Mass spectrometry analysis of phosphatidic acid newly formed in mitochondria revealed that it has been derived from fragmentation of cardiolipin. Thus, free-radical fragmentation of cardiolipin in its polar part with the formation of phosphatidic acid is a likely mechanism that damages mitochondria under conditions of oxidative stress.

Synthesis and Properties of 11-(3,5-Di-tert-butyl-2- hydroxyphenylcarbamoyl)undecanoic Acid, a New Amphiphilic Antioxidant

Based on the membrane addressing concept, designing and synthesis of 11-(3,5-di-tert-butyl-2-hydroxyphenylcarbamoyl)undecanoic acid have been carried out. Antioxidant properties of the prepared compound were investigated in comparison with its non-amphiphilic analogues.

Production of OH radicals in the autoxidation of the Fe(II)–EDTA system

The autoxidation of oxygenated solutions of Fe(II)–EDTA containing dimethyl sulfoxide (DMSO) proceeds with the participation of the OH radical. Its presence as the crucial reactive intermediate has been established by the competition method based on the formation of methanesulfinic acid from DMSO as the reference. As competitors were used guanosine, methanol, tert-butyl alcohol, acetamide, and acetonitrile, whose rate constants span a range of almost three orders of magnitude. In the absence of competitors the yield of methanesulfinic acid is between 1/5 and 1/6 of the Fe(III) formed. Other products are formic acid and formaldehyde. The process of DMSO oxidation is essentially a chain reaction initiated by the OH radical which is at the same time one in a succession of several free-radical chain carriers, and in which the EDTA complex of Fe(II) also participates.

Free radical fragmentation of cardiolipin by cytochrome c.

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H(2)O(2)/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H(2)O(2)/ascorbate or with Cu(2+)/H(2)O(2)/ascorbate.

Influence of lipids with hydroxyl-containing head groups on Fe2+ (Cu2+)/H2O2-mediated transformation of phospholipids in model membranes

Under condition of ROS formation in lipid membranes, free radical reactions can proceed in both hydrophobic (peroxidation of lipids, POL) and polar (free radical fragmentation) parts of the bilayer. Free-radical fragmentation is typical for the lipids containing a hydroxyl group in β-position with respect to an ester or amide bond. The present study has been undertaken to investigate free-radical transformations of phospholipids in model membranes containing lipids able to undergo fragmentation in their polar part. Liposomes from egg yolk lecithin containing saturated or monounsaturated glycero- and sphingolipids were subjected to the action of an HO* - generating system - Fe(2+)(Cu(2+))/H2O2/Asc, and the POL products were investigated. In parallel with this, the effects of monoacylglycerols and scavengers of reactive species on Fe(2+)(Cu(2+))/H2O2/Asc - mediated free-radical fragmentation of phosphatidylglycerols were studied. Hydroxyl-containing sphingolipids and glycerolipids, which undergo free-radical fragmentation under such conditions, manifested antioxidant properties in the model membranes. In the absence of HO groups in the lipid structure, the effect was either pro-oxidant or neutral. Monoacylglycerols slowed down the rate of both peroxidation in the hydrophobic part and free-radical fragmentation in the hydrophilic part of phospholipid membrane. Scavengers of reactive species inhibited the fragmentation of phosphatidylglycerol substantially. Thus, the ability of hydroxyl-containing lipids to undergo free-radical fragmentation in polar part apparently makes a substantial contribution to the mechanism of their protector action.

Radiation-induced free-radical fragmentation of ceramides in a model system

The radiation-initiated transformations of ceramide as a constituent of micelles were studied. An amide was identified as a ceramide γ-radiolysis product. Determination of other radiolysis products and data on the interaction of the FeSO4-H2O2-ascorbate system with ceramide lead to the conclusion that the amide is formed as a result of OH-induced fragmentation in the polar moiety of the substrate.

Ceramide formation on the γ-irradiation of galactocerebroside as a constituent of micelles

The radiation-initiated transformations of galactocerebroside (GalCer) as a constituent of micelles were studied. The radiation-chemical yields of decomposition of the starting substrate and formation of degradation products suggested that the formation of ceramide is the main process in the γ-radiolysis of GalCer. The irradiation of GalCer in the presence of dimethyl sulfoxide, which is an active scavenger of OH radicals, decreased the yield of ceramide. Ceramide was formed as a result of GalCer degradation with O-glycoside bond cleavage in the polar moiety of the lipid.